The invention concerns a DNA coding a eukaryotic highly active alkaline phosphatase with a specific activity of more than 3000 U/mg. Furthermore the invention concerns a process for the production of the DNA according to the invention as well as a vector containing the DNA according to the invention as well as a cell line containing this vector. The invention additionally concerns a recombinant highly active alkaline phosphatase with a specific activity of more than 3000 U/mg which is coded by the DNA according to the invention.
Alkaline phosphatases (AP) are dimeric, zinc-containing, non-specific phosphomonoesterases which are found in all organisms from E. coli to mammals (McComb et al., 1979). Comparison of the primary structure of different alkaline phosphatases showed a high degree of homology (25-30% homology between E. coli and mammalian AP) (Millxc3xa1n, 1988; Harris, 1989).
In humans and higher animals the AP family consists of four members which are coded on different gene loci (Millxc3xa1n, 1988; Harris, 1989). The alkaline phosphatase family includes the tissue-specific APs (placental AP (PLAP), germ cell AP (GCAP) and intestinal AP (IAP)) and the non-tissue-specific APs (TNAP) which are mainly located in the liver, kidney and bones.
A decisive property of the previously known APs is the large variability of the catalytic activity of the mammalian APs which have a 10-100-fold higher specific activity than E. coli AP. Among the mammalian APs the AP from the bovine intestine (bIAP) exhibits the highest specific activity. This property makes the bIAP attractive for biotechnological applications such as enzyme conjugates for a diagnostic reagent or dephosphorylation of DNA. In 1985 Besman and Coleman proved the existence of two IAP isoenzymes in the bovine intestine, the IAP from the calf intestine and the IAP from the intestine of a mature cow (bIAPs), by amino-terminal sequencing of chromatographically purified AP fractions. A clear difference at the amino terminus was described between the bIAP of the mature cow (LVPVEEED) and the bIAP from calf intestine (LIPAEEEN). In 1993 Weissig et al. achieved an accurate biochemical characterization by cloning a recombinant bIAP (bIAP I) with a specific activity of ca. 3000 U/mg and the N-terminus LVPVEEED. However, bIAPs from calf intestine with specific activities of up to 8000 U/mg are also commercially available (Boehringer Mannheim, Biozyme, Oriental Yeast) which, however, have previously not been further characterized. All attempts at cloning these highly active alkaline phosphatases were unsuccessful. It was therefore not possible to produce a recombinant highly active alkaline phosphatase. However, the possibility of recombinant production is absolutely essential for an economic production of highly active alkaline phosphatase.
Consequently the object of the present invention was to provide highly active alkaline phosphatases by recombinant means which can also be cloned. Highly active within the sense of the present invention means that the alkaline phosphatase according to the invention has an at least 10% increased activity compared to previously known alkaline phosphatases.
The object was achieved according to the invention by the provision of a DNA coding a eukaryotic highly active alkaline phosphatase with a specific activity of more than 3000 U/mg, preferably of at least 3500 U/mg in which the amino acid residue at position 322 is smaller than aspartate. A eukaryotic DNA is preferred within the sense of the present invention. Eukaryotic cDNA is particularly preferred which means a DNA that no longer contains introns. The term xe2x80x9camino acid residue smaller than aspartatexe2x80x9d is understood as any amino acid, preferably natural amino acids or amino acids derived therefrom, which has a smaller spatial dimension than the structure of the amino acid aspartate. A DNA according to the invention is preferred in which the amino acid residue 322 is glycine, alanine, threonine, valine or serine. A DNA according to the invention is particularly preferred in which the amino acid residue 322 is glycine or serine. It is quite especially preferred that the amino acid residue 322 is glycine. A DNA according to SEQ ID NO.: 1, 3 and 5 (FIGS. 1,3,5) and the associated amino acid sequence according to SEQ ID NO.: 2, 4 and 6 (FIGS. 2,4,6) are part of the present invention. The present invention also concerns those cDNAs which differ from the afore-mentioned only in that the N-terminus is longer or shorter in comparison to the cDNAs according to SEQ ID NO.: 2, 4 and 6. In such cases the name for position 322 according to SEQ ID NO.: 2, 4 and 6 changes correspondingly. If for example the N-terminus is x amino acids longer or shorter than SEQ ID NO.: 2, 4 and 6, the relevant position 322 is also shifted by x amino acids. SEQ ID NO.: 1 contains the DNA code for the sequence of the highly active bIAPII isoenzyme. The native enzyme was known but not characterized and not possible to clone. Hence the determination of the amino acid sequence of the highly active bIAP II isoenzyme is a subject matter of the present invention. A highly purified fraction with high specific activity from the calf intestine (Boehringer Mannheim) was used to determine the sequence. Peptide maps of the highly active AP were produced by cleavage with the endoproteinases LysC, AspN, GluC, trypsin and chemical cleavage by bromocyanogen. The peptides produced in this manner were separated and isolated by means of reversed phase HPLC. Each peptide was analysed by electrospray mass spectroscopy and sequenced by means of Edman degradation. The sequences obtained in this way were compared with the published sequence of bIAP I (Weissig et al., 1993). As expected the amino terminus of bIAP II has the start sequence LIPAEEEN as described by Besman and Coleman (J. Biol. Chem. 260, 11190-11193 (1985)). The complete amino acid sequence of bIAP II is shown in SEQ ID NO.: 2 (FIG. 2). According to this the bIAP II has a total of 24 amino acid substitutions compared to bIAP I. The number of amino acids in the isolated highly active bIAP II isoenzyme is 480 amino acids. The nucleotide sequence of 1798 bp (FIG. 1) includes a coding region of 514 amino acids. The amino acids that are possible from position 481 to 514 inclusive can vary within wide limits.
In the following the present invention describes the cloning and complete characterization of two new previously unknown bIAPs (bIAP III and bIAP IV). Northern blot analyses were carried out on RNA samples from different sections of the bovine intestine. A cDNA bank of the probes with the strongest hybridization signal was set up with an oligo dT primer (Stratagene, San Diego, Calif., USA) in the vector IZAP II (Stratagene, San Diego, Calif, USA). The complete bank (1.0xc3x97106 recombinant clones) was screened with the 1075 bp HindIII fragment of bIAP I which covers a region from exon I to VIII of the bIAP I gene. 65 Clones were isolated and sequenced. In this process two new bIAPs were identified (bIAP III and bIAP IV) whose characterization is described further below and were neither completely homologous to bIAP I nor to bIAP II. The nucleotide sequences of bIAP III and IV are shown in FIGS. 3 and 5. The sequence differences of bIAPs I IV are shown in FIG. 7. However, none of the new bIAPs has the expected N-terminus LIPAEEEN but rather new previously not described N-termini (see FIG. 7). The cDNA of the two new bIAP isoenzymes was recleaved with appropriate restriction enzymes and inserted by ligation into the CHO expression vector pcDNA-3 (e.g. from the Invitrogen Co. San Diego, Calif., USA). The clones which contained the new bIAP isoenzymes were brought to expression according to the method described by Invitrogen and the isoenzymes were characterized. The expression of a bIAP gene in various hosts is described in WO 93/18139 (CHO cells, E. coli, baculovirus system). The methods, vectors and expression systems described in this document are part of the disclosure of the present application. The present invention in addition concerns the native and recombinant highly active alkaline phosphatases bIAP III and bIAP IV. The alkaline phosphatases according to SEQ ID NO.: 4 and 6 are particularly preferred. CHO cell lines containing the bIAP III and bIAP IV gene were deposited at the DSMZ, xe2x80x9cDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbHxe2x80x9d, Mascheroder Weg 1b, D-38124 Braunschweig (DSM ACC 2349, DSM ACC 2350).
In the following the invention describes the construction of the bIAP II sequence by ligation of mutated and wild-type fragments of bIAP I, III and IV. A series of intermediary intermediate products (L1N8, INT 1, INT 2 and INT 3) was generated by this process which code for functional isoenzymes. In order to construct these intermediary intermediate products a section of the bIAP-cDNA to be modified was cleaved out in each case with appropriate restriction enzymes and replaced by a segment of another bIAP-cDNA containing the desired mutations which possesses compatible ends by digestion with restriction enzymes. Mutations which cannot be introduced by ligation of segments of different bIAP-cDNAs were introduced by site-directed mutagenesis. The mutated fragment was subsequently recleaved with appropriate restriction enzymes and ligated into a like-wise cleaved bIAP-cDNA segment with compatible ends (FIG. 8). The mutations introduced in this manner were subsequently checked by restriction analysis and sequencing.
Hence a subject matter of the present invention is a process for the production of the DNA according to the invention characterized in that mutated and wild-type fragments of the DNA of one or several alkaline phosphatases were ligated. Moreover the present invention concerns a cDNA which codes functional isoenzymes and which is formed as intermediate products during the aforementioned process according to the invention. Additionally the present invention concerns a vector containing the cDNA according to the invention.
A further subject matter of the present invention is a cell line containing the vector according to the invention. Suitable cells are for example eukaryotic cells such as CHO, pichia, hansenula or saccharomyces cerevisiae and aspergillus or prokaryotic cells such as E. coli. E. coli, yeast and CHO cells are particularly preferred. Suitable starting vectors for E. coli strains are for example pTE, pTaq, bPL, pBluescript. Suitable E. coli strains are for example XL1-Blue, HB101, RR1 xcex94 M15, BL21(DE), MC 1000 etc. Suitable pichia vectors are for example pGAPZxcex1 and pPICZxcex1 (Invitrogen, San Diego, Calif., USA). A suitable vector for CHO cell lines is for example pcDNA-3 (Invitrogen, San Diego, Calif., USA). A CHO cell line containing the bIAP II gene was deposited at the DSMZ, xe2x80x9cDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbHxe2x80x9d, Mascheroder Weg 1b, D-38124 Braunschweig (DSM ACC 2348).
The kinetic characterization of the recombinant bIAP I, II, III and IV isoenzymes showed considerable differences with regard to the catalytic properties (FIG. 9). For example bIAP II has a more than 300% increased i.e. more than three-fold higher specific activity (ca. 8600 U/mg) than bIAP I (ca. 2700 U/mg). But also bIAP III and bIAP IV exhibit an approximately 1.8-fold (ca. 4700 U/mg) and about 2.6-fold ( greater than 6700 U/mg) higher activity respectively than bIAP I (FIG. 9) which corresponds to a percentage increase of ca. 170% and 250% respectively. Furthermore there was a considerable measurable difference in the heat stability of the isoenzymes. bIAP I is the most heat stable isoenzyme, the Tm value of bIAP II and III is 7xc2x0 C. lower and the Tm value of bIAP IV is 13xc2x0 C. lower than bIAP I (FIG. 9). The Tm value is understood as the temperature at which a 50% residual activity is measured after an incubation period of 10 minutes.
In the following the invention describes the identification of amino acid residues which influence the specific activity of the bIAPs. This was aided by the intermediary intermediate products. The expression of the intermediary chimers L1N8, INT 1, INT2 and INT3 enabled 11 of the 24 amino acids to be excluded as an effector for the increase in activity (FIG. 7).
The L1N8 mutant enzyme had a comparable specific activity to bIAP I; consequently the mutations V2I, V4A and D8N introduced in this case are not relevant for the increase in the specific activity. The notation V2I means that at position 2 the amino acid valine is replaced by isoleucine.
The INT 1 mutant has a comparable specific activity to bIAP II and consequently this region is important.
The INT 2 mutant has a comparable specific activity to INT 1 and bIAP II and consequently the mutations S380G, D411G, D416E, Q420R, Q427L, E453Q and T480A from INT 2 can also be excluded.
In generating the INT 3 mutants no change in the high specific activity was found thus excluding an effect of the mutation N192Y.
In order to identify which of the 13 remaining residues are crucial for the high specific activity, the bIAP II cDNA was used in the present invention as a template for single mutations against the corresponding amino acid of bIAP I. The single mutants N122K, I133M, A142S, K180M, M205K, E210V, E236A, G322D and I332G as well as a combined A289Q-A294V-Q297R-L299V bIAP II mutant were constructed (FIG. 9).
Surprisingly it was found that mainly the mutation G322D is able to decrease the high specific activity of bIAP II (ca. 8600 U/mg) by more than a factor of 3 (2817 U/mg) and thus to convert it into the comparably low specific activity of bIAP I.
In order to verify this result the reverse mutation D322G was introduced into bIAP I in the present invention. Surprisingly in this case the reverse effect namely an increase of the specific activity of more than 3-fold to 10148 U/mg was measured and hence a comparable value to bIAP II was achieved (FIG. 9). A comparison of the amino acid sequences of the relatively more highly active bIAP III (ca. 4700 U/mg) and the more highly active bIAP IV ( greater than 6700 U/mg) again confirm this result. bIAP III has a serine at position in 322 and bIAP IV has a glycine.
In addition in the present invention the generated mutants were in turn examined for heat stability. Consequently the difference in the heat stability between bIAP I and bIAP II is due to a combined effect of more than one substitution. The [G322]bIAP I as well as the [D322]bIAP II mutants exhibit stability values which lie between those of the bIAP I and bIAP II isoenzymes (FIG. 9). The D322G mutation has a slight destabilizing effect (almost 4xc2x0 C. in T50) on the bIAP I isoenzyme whereas the substitution G322D in bIAP II results in a corresponding increase in the stability of this mutant enzyme. However, the heat stability of the wild-type bIAP I is not achieved.
Hence the subject matter of the present invention is in particular to provide a highly active recombinant alkaline phosphatase with an activity of more than 3000 U/mg which is coded by a eukaryotic cDNA. A highly active recombinant alkaline phosphatase according to the invention is particularly preferred in which a glycine, alanine, threonine, valine or serine is at position 322. An alkaline phosphatase according to the invention is particularly preferred in which a glycine is at position 322.
The highly active recombinant alkaline phosphatase according to the invention can preferably additionally have a mutation at one or several of the following positions:
Amino acid residues at position 1, 108, 125, 149, 181, 188, 219, 221, 222, 223, 224, 231, 252, 258, 260, 282, 304, 321, 330, 331, 354, 383, 385, 400, 405, 413, 428, 431 and 461 in which the mutation causes an increase in activity. Furthermore the present invention concerns a process for the production of the highly active alkaline phosphatase according to the invention. The alkaline phosphatases according to the invention can also be further improved by specific mutagenesis e.g. with regard to their thermostability.
The activity of the highly active alkaline phosphatase according to the invention was determined according to E. Mxc3x6ssner et al., Z. Physiol. Chem. 361 (1980), 543-549; with the difference that the test was carried out at 37xc2x0 C. rather than at 25xc2x0 C. as described in the publication. The determination at 37xc2x0 C. is the world-wide usual temperature at which the activity is measured in diethanol buffer (BM test method 5426).
The protein determination of the APs according to the invention and of the known APs is carried out by measuring the absorbance of the protein solution at 280 nm against water. The absorbance of a low and highly active AP solution at a concentration of 1 mg/ml is 1.0 at 280 nm (A 280 nm (1 mg/ml) equals 1).
The specific activity is determined by forming a quotient of activity relative to the accompanying amount of protein.